Cryopreservation of cells, tissues and organs has vast implications on numerous procedures, for example grafting, in vitro manipulation (such as in vitro fertilization), research, etc. In a conventional slow-freezing method used for biological samples, a chamber is used in which the sample is introduced for freezing. Then, the temperature of the chamber is dropped in a controlled stepwise manner, thus exposing the sample to an external and gradual change in temperature.
A different technology for freezing is the “Multi-temperature gradient” (MTG) directional solidification, which is based on the invention disclosed in U.S. Pat. No. 5,873,254. In this technology, the sample is moved at a constant velocity (V) through temperature gradients (G) so the cooling rate (G×V) and ice front propagation are controlled and the velocity of the movement of the sample determines the morphology of the ice crystals formed within the sample. This method also enables the incorporation of controlled seeding into the freezing process.
The freezing of samples according to any of the known methods, even when using accurate freezing rate control systems, is typically adapted for small samples that are 5 milliliters or less in volume. This is partially due to the fact that, in large samples, some parts of the sample (usually the outer zone or part thereof) may chill or warm faster than other parts. Thus, freezing and storage of semen is performed regularly using mini (¼ cc) or midi (½ cc) straws. Samples with volumes of nearly 5 milliliters are usually frozen in plastic bags that are flattened during the preservation process, so as to have at least one dimension of the sample not exceeding 0.5 cm.
Solutions enabling freezing of larger samples have been suggested in co-pending PCT/IL02/00738 and PCT/IL03/00026. In the former application, a method is described wherein the sample is agitated during freezing under the directional solidification process. Thus, the rate of heat transfer within the sample is amplified, and the effect of the sample's bulk, morphology and heat transfer rate on the morphology of the forming ice crystals is reduced. In the latter application, an “isothermal-break” method is disclosed. In this method, the freezing or thawing of a sample is performed such that the sample is kept at a desired intermediate temperature at least for a time that would allow the temperature of the sample or a section thereof to become uniform and equal to the intermediate temperature.